The overall objective of the proposed work is to optimize the methodology for folate analysis of food employing microbiological assay, by identifying improved procedures in various stages of the assay. The results obtained may lead to standardization of the analytical methodology for folate determination in foods and to stimulate further research that would increase the reliability and validity of food folate database. Accurate food-composition and intestinal-absorption data are essential to further our understanding of the bioavailability of food folate and to assess the human dietary folate requirements. Significant health benefits may also derive from update food folate information in light of the recently acknowledged roles of folate in neural tube defects, cardiovascular disease and cancer. There are three major steps in the microbiological assay for food folate: sample extraction, enzyme treatment and Lactobacillus casei assay. Variations in folate values of selected food items caused by varied conditions in each stage of the analysis will be quantified. The variables in the extraction procedures to be studied are: method of sample homogenization, extracting buffer used, ratio of sample to buffer, completeness of sample extraction, level of ascorbic acid added to buffer, heat treatment, pH adjustment of sample extract, method for clarification of sample extract, and storage stability of sample extract. In enzyme treatment of samples, the type and source of enzymes and conditions for incubation will be examined. Specific variables in the L. Casei assay to be investigated include the source and preparation of the calibrant, preparation of inoculum and culture medium, time and temperature of incubation, volume of sample extract to be assayed and the adaptation of microplate bioanalytical techniques for L. Casei assay. Additional specific objectives are to establish optimal conditions for sample analysis in selected food items and to determine if a general analytical procedure may be applicable to various types of food for folate determination. The final component of the proposed work focuses on the application of the refined analytical methodology to study the kinetics of folate destruction and the retention of folate during food preparation, processing and storage.